In Vitro
B I O S Y N T H E S I S OF M I T O C H O N D R I A L M E M B R A N E P R O T E I N S
In Vitro Biosynthesis of Membrane Proteins in Isolated Mitochondria from Saccharomyces carlsbergensis" Sophia Yangt and R. S. Criddle
:The products of in vitro incorporation of [14C]amino acids into isolated mitochondria from Saccharomyces carfsbergensis have been investigated and found to be associated with the membrane fraction. The yeast mitochondrial membrane proteins were fractionated into four major subfractions. The proteins of these subfractions were then solubilized by reduction and carboxymethylation or sulfonation in the presence of sodium dodecyl sulfate and separated by disc gel electrophoresis. The level of incorporation of radioactive amino acids was rather low; however, it was not due to microsomal or bacterial contamination. Actinomycin D, rifamycin, and ethidium bromide had no effect on this in vitro incorporation into mitochondria. CS-I, the predominant component from ABSTRACT
T
he ability of isolated mitochondria to incorporate radioactive amino acids into protein was first detected by Siekevitz (1952). Later studies by Roodyn et af. (1961), Truman and Korner (1962), Simpson (1962), and Kroon (1963a,b, 1964) have confirmed the original observation; however, solution conditions reported for efficient incorporation were rather conflicting. In recent reports, Wheeldon and Lehninger (1966), Beattie et a/. (1967), and Lamb et a/. (1968) have established quite well the requirements for in vitro amino acid incorporation. Most of the radioactivity incorporated has been shown to be associated with insoluble membrane proteins (Beattie et a/., 1967; Haldar et al., 1966). Haldar et a / . have demonstrated that several protein species separable on disc gel electrophoresis have incorporated radioactive amino acid; however, no specific protein products of mitochonrial synthesis have been resolved. The present studies provide a reproducible method for separation of membrane components by extraction and electrophoretic procedures, demonstrate that one of the membrane protein fractions contains the major product(s) of protein synthesis in isolated yeast mitochondria, and describe some properties of the protein from various membrane fractions. Materials and Methods D-Chloramphenicol, cycloheximide, oligomycin, and bovine pancreatic trypsin (type I) were obtained from Sigma Chem-
* From the Department of Biochemistry and Biophysics, University of California, Davis, California 95616. Received January 21, 1970. This work was supported in part by U. S. Public Health Service Grant GM 1001-17.
t Predoctoral
Trainee of U. S . Public Health training grant. Work done in partial fulfillment for the requirements of Ph.D. degree, University of California, Davis. Present address: Section of Biochemistry and Molecular Biology, Cornell University, Ithaca, N. Y.
membrane fractionation, has the highest specific radioactivity and accounts for 50-60Z of the total radioactivity incorporated into insoluble membrane proteins. The incorporation of radioactive amino acid into CS-1 is strongly inhibited by chloramphenicol. While other membrane components are labeled to a much lesser degree, incorporation into these other components is not particularly sensitive to chloramphenicol inhibition. Comparison of amino acid compositions and fingerprints of peptides obtained from tryptic digestion of the three major membrane components, CS-1, Ci-1, and Ci-3, suggest that all are extremely similar in spite of differences in labeling and in migration in a sodium dodecyl sulfate containing disc gel electrophoresis system.
ical Co. Rabbit muscle A grade pyruvate kinase and ethidium bromide were purchased from Calbiochem. Rifamycin was purchased from Dow Chemical. Actinomycin D was purchased from Merck Sharp and Dohme. Glusulase was obtained from Endo laboratory. [14C]Protein hydrolysate was purchased from Amersham-Searle Corp. [ 4C]Leucine, [ "1valine, [ 14C]lysine,and [ 3H]protein hydrolysate were obtained from New England Nuclear Corp. [14C]Tyrosineand [ I C ] arginine were purchased from Schwartz BioResearch Inc. Other reagents were reagent grade and used without further purification unless otherwise stated. [solation and Sofubifization of Mitochondrial Membrane Components. ISOLATION OF MITOCHONDRIA. A double mutant of Saccharomyces carlsbergensis requiring both lysine and methionine for growth was employed in these studies. Cells were broken either by mechanical means or by enzymic digestion. MITOCHONDRIA PREPARED FROM MECHANICALLY BROKEN CELLS. Yeast cells were grown aerobically at 25-28' (room temperature) in 1 peptone-1 % yeast extract-1 % glucose medium and were harvested in late-log phase when the absorbance at 660 ml.c reached 8-9 optical density units and were collected with a Sharples centrifuge. A suspension of 10 g of cells was added to bottles containing 20 g of 0.05-0.45-mm glass beads and homogenized in a B. Braun cell homogenizer at 0-4' for 45 sec. Crude mitochondria were obtained by differential centrifugation at lOOOg and 17,OOOg. The crude mitochondrial fraction, suspended at 20 mg/ml in STE' buffer, was purified in a 20 % to 60 % (w/w) sucrose density gradient. All sucrose solutions were made in TE buffer. The density
1 STE buffer, 0.01 M Tris-CI-0.25 M sucrose-0.001 M EDTA (pH 7.4); T E buffer, 0.01 M Tris-CI-0.001 M E D T A (pH 7.4); M E T buffer, 0.2 M mercaptoethylamine-0.01 M EDTA-0.1 M Tris-CI (pH 8.0); CPS buffer, 3.08 mM citric acid-21.6 mM Na2HPOd-1 mM EDTA-1.2 M sorbitol (pH 6.8).
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gradient was preformed in a Beckman L-4 zonal centrifuge at 4000 rpm at 4". Equilibrium was attained after centrifuging at 20,000 rpm for 8-12 hr. The purified mitochondrial fractions were pooled, diluted with an equal volume of T E buffer, and centrifuged at 30,000 rpm in the Sorvall ultracentrifuge for 1-2 hr. The mitochondria thus obtained were used in preparative scale isolation of membrane protein subfractions. MITOCHONDRIA PREPARED FROM SPHEROPLASTS. Cells were grown and harvested as above. Spheroplasts were prepared using a modification of the method of D~iellet ul. (1964). Each gram of cells was incubated with 2.5 ml of MET buffer at 30" for 30 min. The suspension was centrifuged at lOOOg for 10 min and the pellet was washed with CPS buffer three times. The final pellet was suspended in CPS buffer (1.7 ml of CPS buffer,'g of cells) and incubated with glusulase (20 mg of enzyme,'g of cells) at 30" for 1 hr. The suspension was centrifuged at lOOOg, washed with CPS buffer three times and centrifuged at 3000g for 10 min. The spheroplast suspension was homogenized in a Potter-Elvehjem glass homogenizer in the lysis buffer (2 ml of lysis buffer,'g of cells) containing 0.4 hi sorbitol20 mM MgCI2-10 m b i KC1-5 mhi phosphate (pH 6.8). The homogenate was mixed vigorously with Vortex mixer and then incubated 20 min with shaking at 0". A mitochondrial pellet was obtained by differential centrifugation and then purified by equilibrium sedimentation in linear sucrose density gradients (Criddle and Schatz, 1969). Mitochondria prepared in this way were used in all [14C]aminoacid incorporation experiments. FRACTIONATION OF MEMBRANE PROTEINS. Purified mitochondrial protein was fractionated initially into subfractions by the method of Richardson et ul. (1966). Soluble mitochondrial protein was removed by freeze and thaw treatments followed by washing the mitochondria in 0.4 M KCI solution. The KC1-insoluble membrane protein was solubilized with deoxycholate (2 mg/mg of protein)-cholate (1 mg;mg of protein) solution and precipitated by addition of saturated (at 4") (NH&SO, solution to a final concentration of 15 % saturation. Lipid was extracted with 95% acetone at 0" and the "structural protein" subfraction thus obtained was washed with 0.01 hf Tris-C1 buffer (pH 8.5) three more times. The product was either washed with 8 M urea (pH 5.5) at 5 mg'ml according to the method of Lenaz and Green (1968) or suspended in 8 M urea-0.1 N NaOH solution and then dialyzed exhaustively against deionized water. All protein fractions were collected and analyzed as described below for level of [14C]aminoacid incorporation. SOLUBILIZATION OF MEMBRANE PROTEIN SUBFRACTIONS Bl' REDUCTION AND CARBOXYRIETHYLATION. Membrane protein sub-
fractions were suspended in 0.01 hi Tris-CI (pH 8.5)-0.5 % SOdium dodecyl sulfate solution to a final concentration of 2 mg,' ml and reduced with 100 X excess (mole!mole of protein of 20,000 molecular weight) of dithiothreitol (20 mhi) for 4 hr at room temperature. A tenfold molar excess of iodoacetate (relative to dithiothreitol) was added and the reaction was allowed to proceed for 1 hr in the dark at p H 8.5 and room temperature, The reduced and carboxymethylated protein was dialyzed against three changes of 0.01 RI Tris-C1 (pH 8.5) buffer. SOLUBILIZATION BY SULFONATION OF \lE\IBRANE
PROTEIN
Sulfonation was carried out essentially as reported by Chen (1968). Membrane proteins were suspended in 1 sodium dodecyl sillfate-0.2 11Tris-CI (pH 8.4) solution
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and then incubated with shaking in a medium containing 0.05 Na2S03,5 X lo-' M cysteine, 2 X lo-' M c u S o 4 , 0.5% sodium dodecyl sulfate, and 0.1 M Tris-C1 (pH 8.5) at 25" for 1 hr. The solution was then dialyzed against 0.1 M Tris-C1 (pH 8.5) with three changes of buffer. FRACTIONATION OF COMPONENTS FROM SOLUBILIZED MITOCHOSDRIAL SUBFRACTIONS BY DISC GEL ELECTROPHORESIS. The system of preparative disc gel electrophoresis published in the Biichler Instrument Manual (Ornstein and Davis, 1964) modified to contain 0.03% sodium dodecyl sulfate in the upper buffer was used to further fractionate the major subfractions into various components. Preparative disc gel electrophoresis was carried out in 0.5 X 3.375 in. tubes with 7 ml of running gel and 1.2 ml of stacking gel solution. Stacking and running were carried out at 4 and 8 mA per tube, respectively. Protein bands were identified by staining gels in 0.1 Buffalo blue black in 40 % ethanol-15 % acetic acid solution for 15 min, destaining with 10% acetic acid until gels were almost completely clear and then washing in 40 ethanol-5 acetic acid solution. The gels were scanned in a Schoeffel spectrodensitometer SD 3000 at 540 mp. Protein bands could also be detected by cooling the gels to 0" for 4 hr. Protein-bound sodium dodecyl sulfate precipitated in the cold and facilitated the identification of protein bands. ISOLATION OF MEMBRANE COMPONENTS. The major membrane components separated by preparative disc gel electrophoresis were located by cold precipitation of the sodium dodecyl sulfate-protein bands and the gel segments cut out accordingly. The membrane components were pooled and eluted electrophoretically through a coarse sintered glass filter and into attached dialqsis tubing. The protein components were dialyzed against three changes of 0.01 N Tris-C1 (pH 8.5) buffer. Cliaracterizurioti o f Various Metribrune Components. REhlOVAL OF PROTEIN BOUND SODIUM DODECYL SULFATE. Membrane components were treated with 0.5 N K O H and centrifuged repeatedly to remove most of the bound sodium dodecyl sulfate and then dialyzed exhaustively against deionized water prior to any chemical or enzymatic analysis. MOLECULAR WEIGHT DETERRIINATION. Molecular weights Of' CS-I, Ci-1, and Ci-3 were estimated in sodium dodecyl sulfate gels according to the method of Shapiro et al. (1967). The following proteins with known molecular weight : bovine hemoglobin, ovalbumin, myoglobulin, lactate dehydrogenase, /3lactoglobulin, lysozyme, and chymotrypsinogen A were sulfonated or reduced and carboxymethylated at concentrations of 2 mg lml as described above. Electrophoresis was run in 12 polyacrylamide gel. AMINOACID ANALYSIS. Purified membrane components were h>drolyzed under vacuum in sealed vials for 12, 24, 48, 96 or 24, 48, and 72 hr at 110" in constant-boiling HCl (5.7 K). After evaporating to dryness twice to remove hydrochloric acid. the amino acids were dissolved in p H 2.2 citrate buffer. The analyses were carried out on a Phoenix Precision Instrument Co. amino acid analyzer Model K 800 V G at a temperature of 50' using the buffer system of Moore et a/.(1968). FINGERPRINTING. The sodium dodecyl sulfate free SUIfonated coniponents were suspended in lo-? M N H ~ H C O B lo-; hi CaCly solution at p H 8.5 to 1-2 mgiml and trypsin suspended in .R.I HC1 was added at 0.05 protein concentration. The digestion was carried out at 37" for 6 hr and the reaction was stopped by addition of 25 p1 of 1 N HCl'ml. The peptides were lyophilized, resuspended in H?O, lyophilized, suspended M
z
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BIOSYNTHESIS
OF MITOCHONDRIAL MEMBRANE PROTEINS
in minimum amount of N HCI, and spotted o n Whatman No. 3MM paper. Two-dimensional peptide maps of components were prepared using the procedure of Katz et al. (1959). Chromatography was carried out at room temperature in freshly prepared butanol-acetic acid-water (4:1:5, v/v) for 21 hr. Electrophoresis was run in pyridine-acetic acid-water buffer (25 :1:225, v/v, p H 6.4) for 2 hr in the direction perpendicular to the direction of chromatography. The peptide spots were detected by spraying with 0.3 ninhydrin suspended in 95 ethanol. Biosynthesis of' Mitochondrial Membrane Proteins. INCORPORATION OF ['4C]AMIN0 ACIDS INTO MITOCHONDRIA. Mitochondria prepared from spheroplasts and purified in 20-60 % wjw sucrose gradient were used in all incorporation experiments. The incubation medium used for amino acid incorporation into isolated mitochondria was a modification of that described by Lamb et al. (1968). The standard incubation mixture contained 40 mM Tris-C1 (pH 7.4), 250 mbi sorbitol, 10 mM MgC12, 100 mM KCI, 5 mbi P-enolpyruvate, 1.5 mM ATP, 5 mM PO4 (pH 7.4), and 0.18 mM cycloheximide. Also, each 1 ml of medium contained [14C]amino acid (0.8-1.2 pCi), 10 pg of oligomycin, and 1-3 mg of mitochondrial of protein. A single [14C]amino acid or several different [14C]amino acids in various combinations were used. In the incubation mixtures containing chloramphenicol, the concentration of chloramphenicol was 5 mM. Where possible solutions used were sterilized and operations were carried out in a sterile hood to minimize bacterial contamination. All incubations were carried out at 32" in a metabolic shaker. Mitochondrial suspensions were incubated with protein synthesis inhibitors for 15 min prior to the addition of radioactive amino acids. Incorporation was stopped by addition of an equal volume of cold buffer (0') containing 200 X excess of unlabeled amino acids in STE buffer. After washing three times with STE buffer containing unlabeled amino acids, mitochondrial proteins were fractionated according to the simplified scheme of Figure 1. Aliquots of each fraction were used in counting of radioactivity. The proteins were washed according to the method reported by Criddle and Schatz (1968) and radioactivity of the protein solutions was counted i n ' NuclearChicago scintillation counter using Bray's scintillation fluid. Protein concentrations were determined by the method of Lowry et a/. (1951).
x
x
Mitochondria \Vas11 with 0.4 s IiCl three times (freeze a n d than)
7 Sol~hleprotein (discard)
I
KCI-insoluble membrane protein (100%) Sodium deoxycholate (2 mg/mg of protein) and CHO (1 mg/mg of protein)
I
Sodium deoxycholate insoluble (33 %) Soluble prbtein (67 %) I
Soluhiliie and separate hq disc gel electrophorcsis FIGURE 1 : Scheme for preparation of mitochondrial protein subfractions. The deoxycholate-cholate-insoluble material was fractionated further using the same conditions as used for structural protein fraction. Abbreviations used: SP = structural protein fraction; Dp = deoxycholate-cholate-insoluble fraction; SSP = sulfonated structural protein fraction; SDp = sulfonated Dp fraction.
fractionation of mitochondrial membrane proteins and yield at each step are shown in Figure 1. The sulfonated membrane protein subfractions are readily soluble at low protein concentration. When the SSP subfraction is subjected to disc gel electrophoresis at p H 8.9 in 0 . 0 3 x sodium dodecyl sulfate, four distinctly resolved bands (plus trace amounts of other components) are observed (Figure 2). CS-1, the fastest migrating component, is 45% of the total protein of this fracResults tion; the remainder of the material is distributed among the Isolation of' Mitochondrial Membrane Coinponents. ISOLA- bands Ci-1 (30%), Ci-3 (1273, and Ci-2 plus Ci-4 (10%). TION OF MITOCHONDRIA. The yield of mitochondria per cell Disc gel electrophoresis patterns of the SDp subfraction is also when prepared from spheroplasts is much higher (five times) shown in Figure 2. While similarities exist among the major than the yield when prepared by mechanical breakage of yeast components of these two subfractions, SDp has several additional minor components. The gel patterns of only two subcells. To obtain a high yield of spheroplasts a higher concentration of mercaptoethylamine and higher p H than described fractions are illustrated in Figure 2 ; however all major memin the procedure of Duel1 et a/. (1964) was employed. Lysis of brane subfractions were collected, sulfonted, and separated by spheroplasts in this case becomes the major limitation in obdisc gel electrophoresis. taining high yield of mitochondria. Vigorous stirring at 0' ISOLATION OF MEMBRANE COMPONENTS. Membrane compofacilitates release of mitochondria from the spheroplasts. nents from the CMSP or SSP subfractions were isolated for Mitochondria obtained by this method are more intact than further characterization using preparative scale disc gel electhose obtained by mechanical breakage and form a distinct trophoresis. CS-1, (3-1, and Ci-3 obtained under these condisharp band in sucrose density gradient. tions appear to be pure when protein is reapplied in 8 % gel and FRACTIONATION OF MEMBRANE PROTEINS. The scheme for electrophoresed at p H 8.9 (Figure 3). However, when electro-
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4: Disc gel electrophoresis pattern of CS-I, Ci-I, and Ci-3 (left to right) obtained from SSP subfraction. Electrophoresis was carried out at 12Z gel concentration.The dye front is apparent from marker wire; 80 pg of protein was applied per sample.
FIGURE
2: Disc gel electrophoresis patterns of SSP (a) and SDp (b) subfractions. SSP and SDp (700 pg) were applied on 8Z gel. The front is marked by a copper wire. RGURE
phoresis is carried out at 12x gel concentration, CS-1 separates into four poorly resolved bands termed CS-I a, b, c, and d, while Ci-1 and Ci-3 still migrate as single components (Figure 4). Ci-1 and Ci-3 remain as single gel components at 10-16x gel concentrations (Figure 5). The multiplicity of CS-1 bands seems to indicate either heterogeneity or a process of association and dissociation in the solvent system employed. Disc gel electrophoretic separation at other p H s , i.e., 7.5, 8.3, 11.3, have been attempted, but have not been successful because of protein precipitation or lack of resolution.
D l STA N C E PIGURE 3: Densitometer tracings of disc gel electrophoresispatterns of CS-I, Ci-I, and Ci-3 obtained from SSP subfraction. Tracing with peak at left is Ci-3, center Ci-I, and right CS-I. Electrophoresis was carried out at 8% eel concentration. The dve front is marked by an arrow and the%&tion of migration is lefi to right; 80 pg of each protein was applied.
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Characterization of Membrane Components. MOLECULAR The relative mobilities of standard proteins of known molecular weight as well as of CS-I, Ci-1, and Ci-3 were measured and plotted as relative mobility us. the logarithmic value of the molecular weight. A best-fitted straight line is obtained from this plot. With the exception of the standards lysozyme and lactate dehydrogenase, which show deviation, the other protein molecular weights fall within + 5 % of a straight-line function. Molecular weights of CS-la, CS-lb, CS-lc, CS-ld, Ci-1, and Ci-3 determined according to this method are 15,000, 20,000,26,500,40,000, 39,000, and 56,000, respectively. AMINOACID ANALYSIS. Acid hydrolyses of CS-1, Ci-1, and Ci-3 were run using three to four different hydrolysis times for each protein. Serine and threonine concentrations shown in Table I were determined by extrapolation to zero-time hydrolysis. The amounts of the other amino acids reported were determined at hydrolysis time which ensured complete hydrolysis of the protein. In order to facilitate the comparison of the amino acid compositions in each protein component, results are presented on the basis of an assumed molecular weight of 20,000 and the number of residues of each amino acid in the various components is normalized accordingly. It is apparent that the amino acid composition of all three components, shown in Table I, are extremely similar. On close examination, one notices that Ci-3 has three to four more residues of Glu, one to two residues of fewer Leu, one residue less of Tyr, one to two fewer residues of Lys, and two fewer met residues than either Ci-1 or CS-1. The differences between CS-1 and Ci-1 are indeed minute and can only be detected in Ser and Lys. The number of residues of cysteine and tryptophan of these components have not been determined. FINGERPRINTING. A composite peptide map of the SSP suhfractions is shown in Figure 6. The number of peptides corresponds fairly well with the number of lysine and arginine groups WEIGHT DETERMINATION.
In vitro
TABLE I:
BIOSYNTHESIS OF MITOCHONDRIAL MEMBRANE PROTEINS
Amino Acid Composition of Membrane Compo-
nents:
ASP Thr Ser Glu Pro G~Y Ala Val Met Ile Leu Tyr Phe LYS His Arg NHa 0
cs-1
Ci-1
Ci-3
15 10.8 12.5 16.5 8.75 14.5 15.0 11.7 3.35 10.6 18.6 5.4 8.8 13.8 2.8 7.2 4.73
15 10.8 16.3 15.0 8.5 16.8 16 11.4 3.6 10.1 18.0 5.5 8.9 12.5 2 6.6 20.63
15 10.8 14.5 19.8
i
6.5 15.5 15.6 12.5 1.5 9.5 16.7 4.4 6.5 11.7 2.6 7.7 20.65
The number of residues of all amino acids are normalized
in such a way that the molecular weight of all three components is set at 20,000. Data represent values calculated from 18, 7, and 7 amino acid analyses of CS-1, Ci-1, and Ci-3, respectively.
in the protein obtained from amino acid analysis assuming the molecular weight of the protein to be 20,000. The number of peptides in Ci-1 and Ci-3 is somewhat smaller than in CS-I as seen in Figure 6. Ten peptides are common to all the membrane components studied. In Vifro Biosynthesis of’ Mifochondrial Membrane Proteins. Mitochondria prepared from spheroplasts were used immediately after preparation in all studies of in uitro biosynthesis. The radioactivity from “C-labeled amino acid mixture incorporated into mitochondrial protein as a function of time is shown in Figure 7. Radioactivity is incorporated most rapidly in the first 5 min, and the rate of incorporation levels out slowly and approaches zero after a period of 1 hr. At various time periods during the incubation, 0.1 ml of incubation mixture was plated on enriched agar plates. The number of bacteria in these plates was subsequently counted after incubating the plates at 37’ for 48 hr. The number of bacteria counted was not proportional to the duration of incubation with “C-radioactive amino acids and the maximum number of bacteria detected per milliliter of solution was never greater than 10, thus eliminating bacterial contamination as a major contributing source of incorporation of [“Clamino acids into mitochondria in these experiments. Studies with protein synthesis inhibitors verify this conclusion. Cycloheximide, a specific cytoplasmic protein synthesis inhibitor for eucaryotic cell, was shown to have no effect on [“Clamino acid incorporation into mitochondria. Even though incorporation experiments were routinely run with added cycloheximide, the possibility of incorporation being due to microsomal contamination was investigated by adding
Disc gel electrophoresispatterns of Ci-l and Ci-3 isolated from SSP subfractions. Electrophoresis was carried out at 10, 12, 14, and 16% acrylamide gel concentrations (left to right). The dye front is marked by wire; 100 #g of protein was applied on each gel.
FIGURE 5 :
different concentrations of microsomal material to the incubation mixture. The specific radioactivity incorporated decreased as the concentration of microsomal fraction increased, possibly as a result of nuclease activity. This indicates clearly that the incorporation cannot be accounted for microsomal contamination. The incorporation of [‘Clamino acid into mitochondria is sensitive to osmolality. At 200 mM sorbitol and 32 m M Tris-CI concentration, the protein-synthesizing system has maximal activity.
TABLE 11: Effect of Antibiotics on Amino Acid Incorporation into Isolated Mitochondria.
Added Antibiotics
cpmh
None. Ethanol (50 fig) (1.67%) Actinomycin D (10 fig/ml) Actinomycin D (40 fig/ml) Actinomycin D (80 fig/ml) Rifamycin (5 figlml) Rifamycin (20 fig/ml)
248 303 285 363 342 219 254
Noneb Ethidium bromide (10 f i ~ ) Ethidium bromide (20 f i ~ l Blank
141 151 146 19
each 1 ml of reaction mixture contained 1.0 fiCi of [“C]amino acid mixture. The mitochondrial protein concentration was 0.8 mg/ml. *Each 1 ml of reaction mixture contained 0.8 fiCi of [“Clamino acid mixture. The mitochondrial protein concentration was 0.7 mg/ml.
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ELECTROPHORESI s , I500 VOLTS,
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6: A representative tracing of “fingerprints” of the tryptic digested membrane components. Conditions for analysis are described in Materials and Methods.
FIGURE
Actinomycin D has no measurable effect on the incorporation of [‘Clamino acid mixture into intact isolated mitochondria or osmotically shocked mitochondria (Table 11). Aside from the slight stimulating effect of low concentrations of ethanol added with the actinomycin D, the radioactivity incorporated in the presence of actinomycin D is essentially the
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7 : Time course of [14C]aminoacid incorporation into mitochondria. Each 1 ml of reaction mixture contained I pCi of [14Cc]amino acid mixture, 10 kg oligomycin, and 2.0 mg of mitochondrial protein. At each time interval noted, 2-ml aliquots were removed and the reaction was stopped immediately with 2 ml of cold 10% trichloroacetic acid. FIGURE
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same as the control. Similarly rifamycin, a bacterial RNA polymerase inhibitor, and ethidium bromide, a DNA-polymerase inhibitor, exerted no effect on the incorporation. I u order to clarify whether lack of inhibition by actinomycin D is due to inability of the antibiotic to bind to niitochondrial DNA or whether simultaneous synthesis of mRNA is not required for the protein synthesis in isolated mitochondria under the present experimental conditions, the incorporation of tritiated uridine into shocked mitochondria was investigated in parallel with in citro protein biosynthesis. The results shown in Figure 8 indicate that the incorporation of [3H]uridineinto RNA is 90% inhibited by actinomycin D a t concentrations as low as 10 pcg’ml. Simultaneous incorporation of [14CC]aminoacids is totally unaffected (Figure 9). Rifamycin does not inhibit the initial incorporation of [.’HIuridine into RNA. INCORPORATION OF LABELED AMINO ACIDS IKTO M E M B R A N L PROTEINS. The uniformity of tritium label in all proteins from cells grown in the presence of [”lamino acids serves 21s an internal measure of protein concentration and thus allows ?ICcurate evaluation of specific radioactivity in different membrane components by determination of C::’H ratios. The results of radioactivity incorporated into major mitochondrial subfractions are shown in Table 111. The specific radioactivity increases as the soluble enzymes are removed and the mem-
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TABLE 111: Distribution of
OF MITOCHONDRIAL
MEMBRANE PROTEINS
Radioactivity Incorporated into Major Mitochondrial Subfractions:
Whole mitochondria KCI insoluble Deoxycholate insoluble Ammonium sulfate pellet Sulfonated SP fraction Sulfonated D p fraction
+ Cycloheximide
Chloramphenicol
Cycloheximide 3H
1 4 c
4C/mg
3H
l4C
I4C/mg
2721 1378 2300 731 2878 2532
685 355 5 84 240 1394 878
202 207 204 264 389 279
3321 327 2469 807 2314 4046
281 277 292 24 175 901
68 68 95 24 61 180
zInhibn 66.4 67.2 53.4 90.8 84.4 35.5
a [3H]Amino acid mixture (50 pCi) was added to 1 1. of growing culture of S. carlsbergensis 2.5 generations prior to harvesting the cells. [ 14C]Amino acid was then incubated with isolated mitochondria. For each I-ml incubation mixture containing 1.67 pCi of [I4C]amino acid mixture and 0.1 pCi of [14C]Leu.The mitochondrial protein concentration was 3 mg/ml. Data represent measured counts per minute of 3H and 14C.Ratios of 14C/mgof protein were determined from independent measurement of 3H/mg of protein; 823 cpm of 3Hwas found per mg of protein.
brane proteins are purified in the fractionation process. SSP and SDP subfractions contained the highest specific activity. This incorporation is sensitive to inhibition by chloramphenicol but as shown, not by cycloheximide. While chloramphenicol inhibition noted with this yeast strain is somewhat less than reported with other mitochondrial systems, it is apparent that inhibition of all subfractions occurs and that inhibition is greatest in the SP subfraction. The sulfonated SP and D p subfractions were subjected to disc gel electrophoresis in 12 % gel solutions to determine the
specific activity of the separated CS-1 subbands. The amounts of radioactive labels in each of the proteins of the SSP subfractions are shown in Table IV. The specific activities of CS-1 components are higher than the Ci components. The inhibition of CS-1 by chloramphenicol is also greater than that of the Ci components. The distribution of the radioactivity in different components of the sulfonated D p subfraction is shown in Table V. The specific activities of membrane components in this subfraction are generally somewhat lower than the SP subfraction and the inhibition by chloramphenicol is not as pronounced. However, the CS-1 components are again labeled to a greater extent and their synthesis is affected by chloramphenicol more severely than the Ci components. In Ditro BIOSYNTHESIS OF MEMBRANE PROTEINS IN ISOLATED MITOCHONDRIA OF GLUCOSE-REPRESSED CELLS. The yield of cells of S. carlsbergensis grown in 5 z glucose medium is higher than those grown in 1 glucose; however, the yield of functional mitochondria is greatly reduced. Mitochondria isolated from these repressed cells have the same buoyant density as the functional mitochondria even though the enzymic composition is drastically different. The white color of mitochondria obtained is indicative of the low content of cytochromes. The kinetic profile for the incorporation of a [14C]amino
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8: Time course of [3H]uridine into osmotically shocked mitochondria in the presence or in the absence of inhibitors. Each 1 ml of reaction mixture contained 1.85 kCi of [aHIuridine or 0.8 pCi[14C]aminoacid mixture, 10 pg of oligomycin, and 1.5 mg of mitochondrial protein. At each time interval noted, 1.5-ml aliquots were removed and the reaction was stopped immediately with 1.5 ml of cold 10% trichloroacetic acid. (0-0) Control; (A-A-A) rifamycin. 10 pglml; ( X - - X - - X ) actinomycin D, 1 pg/nil; (0-0) actinomycin D 10, pg/ml. FIGURE
0 0
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20
30
TIMEMIN
9 : Incorporation of [14C]amino acids into mitochondria in the presence of inhibitor. of RNA production. Reaction conditions and definition of points on curve are described in Figure 8.
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~~
Distribution of Radioactive Label in Proteins of Sulfonated Structural Protein Subfraction TABLE IV:
2
Cycloheximide
ci-l Ci-2 CI-3 ci-4 CI-5
"C
3H
rng
I4C
3H
597 406
1032 825 557 554 1505
477 407 823 396 44 172 74 120 100
26 32 26 5 4 47 33 44 32
1101 887
553
265 81 218 267 227 161
1045
2740 1570 1355
6lY
533 1859 10g.5
2284 1301 1201
D;iti rcpresents measured cpc for W a n d itionof ' Y i i i g ~ v ~ i i ~Talde111 t~in
'.'C mg
20 30 35 7 2 37 12 28 22
v : Distribution of Radioactive Label In Protein5 of
Sulfonated Deoxycholate-Insoluble Subfract ton
Chloramphenicol -k Cq cloheximide
'"/
CS-1A CS-IB CS-1C CS-ln
TABIE
Cycloheximide
74 Inhthn 95 8 92 7 95 8 98 Y5
78 5 83 76 h 7x
I'C
CS-I A 23 1 CS-1B 247 CS-IC 332 cs-ln 274 317 Ci-1 CI-2 21 I CI-3 99 ct-4 166 Ct-5
6h
'H
471 672 162 656 1763 I320 1315
1137 1226
' 4C
Cqclohexiniide Chlor'imphenicol
nig
'V:
405 304 360 344 148 132 62 121 45
39 54 16
35 216 82 76 67 89
' 'C It1
tng
548 694 597 630 2017 1058 1483
59 65 22 46 86 65 42 51 17
1079 1 Sh8
"/, Tnhihn
85 4 78 6 94 67 42 51 32 58 0
Deternil-
Data represents nieasured cps for 3H and 1 T . Ikterinination o f 14C!mgwxsnx itiTnbleT11.
acid iiiixtiire into protriti reseinhlcs those of functional mitochondria with its rate decreasing t o x r o i n 30 mill; tiowrwr the level of the incorpor;itioti is only 10% o f the incorporation into nor ina 1 mi t o c ho n t l r i I I . The c1tiantitatii.e distribution o f proteins fractionated into \ ;trioti\ iiiciii1,raiie cotnponcntc ic also difrerent from thi: nor rna I I11i t o rh o ti d r i ii . A p pi-ox i ti iii t cIy 70 06 of the ii I c in b rii ne prott'in is not s?liihilircd h q dcoxqcholatc and cholatc soluL i o i i y . I licr.t>fcvt>. thi. ;iiiiourit o f thr SP tiitvnhranc suhfraction gr t a l l ) rcdiiccil. When tlit. stiIfon;i~ctl$1' a n d Op sithfriiction\ \vcrc sul>.iectid to elcctrophorcsis in thi:, case. 30 I:; o f the total protein rtwiained a t the interface between the running gel a n d stacking gels. Also. the distribution of the viiriotis components is dilfcrent from that obsercetl in fttnctional mitoihonclria. CS-I accounts for only 20% of thc protein in the SSPor in the SDp suhfractions. 7 he distributions of [ LIC']amino a c i d incorporated into various components o f the i,cpressed niitochondriti is i i l i o altered. It is notid that ii gel component migrating at the position of Ci-21 has the highest specific activity and its synthesis is inhibited most by chloramphenicol. Since the incorporation level is low. the a1x)lute ~iingiiittideof the incorporation is not certain; however, it is clear the repressed mitochondria behave diferently froni the normal mitochondria in their ;ihilit> t o synthesize membrane protein components.
thc hest resolution of the mcinbrane component\. In contrast i o prcl iously r q m t e d systetm (Lenaz and Green, 1968 ; 1 ak:iyama r / d., 1966; Le.isek a n d Lusena, 1969). 1007;; o f ttic protisin prcparation enters the gcl a n d its distribution c a n be quantitatively evaluated. Whcn lowcr pH hulTer systems arc LISLYI, ;I high percentage o f protein tloes not penetrate t h c gel, h u t instcxl remains at the interface of the large pore s i x gel. With the highcr pH sqstems employed, ii fast-migrating Ixintl is ohserved hiit other. h a n d \ arc not well resolvt~tl. Of all the mcnihranc coniponents isolated. C'S-I is the tiiiijor ni
